Summary
Square Bin
Cell Bin
Image

Spatial Gene Expression Distribution

Spatial gene expression distribution plot shows MID count at each spot (bin200)

Tissue Plot with Spot (bin200) Colored by MID Count

1,667,882,652

Total Reads

71,895

Mean MID per Bin200

8,182

Mean Gene Type per Bin200

135,184,151

Unique Reads

Key Metrices

Total Reads

Total number of sequenced reads

Valid CID Reads

Number of reads with CIDs matching the mask file and with MIDs passing QC

Invalid CID Reads

Number of reads with CIDs that cannot be matched with the mask file

Discarded MID Reads

Number of reads with MID that have been discarded since MID sequence quality does not satisfy with further analysis

Clean Reads

Number of Valid CID Reads that have passed QC

Non-Relevant Short Reads

Number of non-relevant short reads

Uniquely Mapped Reads

Number of reads that mapped uniquely to the reference genome

Multi-Mapped Reads

Number of reads that mapped more than one time on the genome

Unmapped Reads

Number of reads that cannot be mapped to the reference genome

Transcriptome

Number of reads that are aligned to transcripts of at least one gene

Unannotated Reads

Number of reads that cannot be aligned to transcript of one gene (intergenic)

Unique Reads

Number of reads in Transcriptome that have been corrected by MAPQ and deduplicated

Sequencing Saturation

Number of reads in Transcriptome that have been corrected by MAPQ with duplicated MID

Total Reads

100%

1,667,882,652

Valid CID Reads

81.0%

1,350,879,255

Clean Reads

88.6%

1,196,659,656

Uniquely Mapped Reads

75.9%

908,756,317

Transcriptome

89.5%

813,621,971

Unique Reads

16.6%

135,184,151

Sequencing Saturation

83.4%

678,437,820

Unannotated Reads

10.5%

95,134,346

Multi-Mapped Reads

19.4%

232,616,185

Unmapped Reads

4.6%

55,287,154

Non-Relevant Short Reads

11.4%

154,219,599

Invalid CID Reads

15.4%

256,484,063

Discarded MID Reads

3.6%

60,519,334

Information

Species

mouse

Tissue

kidney

Reference

mouse

FASTQ

V350195543_L02_read_1 V350195543_L04_read_1
V350195543_L01_read_1 V300116245_L02_read_1
V350195543_L03_read_1

Sequencing

Total Reads

Total number of sequenced reads

Total Q30

Ratio of bases with quality value exceeded 30 in the sequenced reads

CID Q30

Ratio of CID (Coordinate ID) bases with quality value exceeded 30

MID Q30

Ratio of MID (Molecule ID) bases with quality value exceeded 30

Total Reads

1,667,882,652

Total Q30

92.44%

CID Q30

89.6%

MID Q30

84.13%

Annotation

Exonic

Number of reads that mapped uniquely to an exonic region and on the same strand of the genome

Intronic

Number of reads that mapped uniquely to an intronic region and on the same strand of the genome

Intergenic

Number of reads that mapped uniquely to an intergenic region and on the same strand of the genome

Transcriptome

Number of reads that mapped to a unique gene in the transcriptome. These reads are considered for MID counting. (Transcriptome = Exonic + Intronic)

Antisense

Number of reads mapped to the transcriptome but on the opposite strand of their annotated gene

Exonic

780,161,468

Intronic

33,460,503

Intergenic

95,134,346

Transcriptome

813,621,971

Antisense

126,433,296

RNA Mapping

Clean Reads

Number of Valid CID Reads that have passed QC

Uniquely Mapped Reads

Number of reads that mapped uniquely to the reference genome

Multi-Mapped Reads

Number of reads that mapped more than one time on the genome

Unmapped Reads

Number of reads that cannot be mapped to the reference genome

Clean Reads

1,196,659,656

Uniquely Mapped Reads

908,756,317

Multi-Mapped Reads

232,616,185

Unmapped Reads

55,287,154

Tissue Segmentation

Tissue

DNB Under Tissue

Number of DNBs under tissue coverage region

mRNA-Captured DNBs Under Tissue

Number of DNBs under tissue that have captured mRNA

Genes Under Tissue

Number of detected gene under tissue coverage

Number of MID Under Tissue Coverage

Number of MID under tissue coverage

Fraction MID in Spots Under Tissue

Fraction of MID under tissue over total unique reads (MID Under Tissue / Unique Reads)

Reads Under Tissue

Number of reads with position prior to filtration under tissue coverage

Fraction Reads in Spots Under Tissue

Fraction of mapped reads under tissue over total mapped reads (Mapped Reads in Spots Under Tissue / (Valid CID Reads + Discarded MID Reads))

DNB Under Tissue

60,052,125

mRNA-Captured DNBs Under Tissue

33,787,671

Genes Under Tissue

25,633

Number of MID Under Tissue Coverage

115,176,890

Fraction MID in Spots Under Tissue

85.20%

Reads Under Tissue

1,165,272,042

Fraction Reads in Spots Under Tissue

82.56%

Sequencing Saturation

(left) Plot of the sequencing saturation as a function of sequencing depth(bin200)

(middle) Plot of the median number of genes detected per bin as a function of sequencing depth(bin200)

(right) Plot of the unique reads detected per bin as a function of sequencing depth(bin200)

Tissue Square Bin Statistics

Bin Size

The size of Bin which is the unit of aggregated DNBs in a squared region (i.e. Bin 50 = 50 * 50 DNBs)

Mean Reads (per bin)

Mean number of sequenced reads divided by the number of bins under tissue coverage

Median Reads (per bin)

Median number of sequenced reads divided by the number of bins under tissue coverage (pick the middle value after sorting)

Mean Gene Type (per bin)

Mean number of unique gene types divided by the number of bins under tissue coverage

Median Gene Type (per bin)

Median number of unique gene types divided by the number of bins under tissue coverage

Mean MID (per bin)

Mean number of MIDs divided by the number of bins under tissue coverage

Median MID (per bin)

Median number of MIDs divided by the number of bins under tissue coverage

Bin Size

Mean Reads
(per bin)

Median Reads
(per bin)

Mean Gene Type
(per bin)

Median Gene Type
(per bin)

Mean MID
(per bin)

Median MID
(per bin)

27.71

2.37

3.41

7,710

7,565

412

421

762

749

47,714

48,307

1,807

1,915

4,716

4,738

100

187,342

193,020

4,418

4,793

18,517

18,885

150

414,982

436,915

6,577

7,196

41,017

42,498

200

727,385

769,553

8,182

8,981

71,895

75,114

Bin 20

(left) Scatter plot of MID count and gene types in each bin

(right) Violin plots show the distribution of deduplicated MID count and gene types in each bin

(bottom) Univariate distribution of MID count, gene types, and DNB numbers with rug along the x axis

Bin 50

(left) Scatter plot of MID count and gene types in each bin

(right) Violin plots show the distribution of deduplicated MID count and gene types in each bin

(bottom) Univariate distribution of MID count, gene types, and DNB numbers with rug along the x axis

Bin 100

(left) Scatter plot of MID count and gene types in each bin

(right) Violin plots show the distribution of deduplicated MID count and gene types in each bin

(bottom) Univariate distribution of MID count, gene types, and DNB numbers with rug along the x axis

Bin 150

(left) Scatter plot of MID count and gene types in each bin

(right) Violin plots show the distribution of deduplicated MID count and gene types in each bin

(bottom) Univariate distribution of MID count, gene types, and DNB numbers with rug along the x axis

Bin 200

(left) Scatter plot of MID count and gene types in each bin

(right) Violin plots show the distribution of deduplicated MID count and gene types in each bin

(bottom) Univariate distribution of MID count, gene types, and DNB numbers with rug along the x axis

Clustering

(left) Clustering spots (bin200) under tissue covered with the Leiden algorithm

(right) UMAP projections of spots (bin200) colored by automated clustering. Same color is assigned to spots that are within shorter distance and with similar gene expression profile

Tissue Plot with Spots (bin200)

UMAP Projection of Spots (bin200)

Cell Bin Statistics

Cell Count

Number of cells

Mean/Median Cell Area

Mean/Median cell area (pixel)

Mean/Median DNB Count

Mean/Median number of DNBs that have captured-mRNAs per cell

Mean/Median Gene Type

Mean/Median gene types per cell

Mean/Median MID

Mean/Median MID count per cell

Cell Count

110,976

Mean Cell Area

414

Median Cell Area

355

Mean DNB Count

242

Median DNB Count

204

Mean Gene Type

435

Median Gene Type

375

Mean MID

836

Median MID

655

Cell Bin

(left) Scatter plot of MID count and gene types in each bin

(right) Violin plots show the distribution of deduplicated MID count and gene types in each bin

(bottom) Univariate distribution of MID count, gene types, DNB numbers, and cell area (pixel) with rug along the x axis

Clustering

(left) Clustering spots (cell bin) under tissue covered region with the Leiden algorithm

(right) UMAP projections of spots (cell bin) colored by automated clustering. Spots in the short distance are assigned with the same color have similar gene expression profile.

Tissue Plot with Spots (cell bin)

UMAP Projection of Spots (cellbin)

Image Information

Manufacture

motic

Model

Moticam Pro 500i

Scan Time

2023-7-17 16:30:0

Overlap

0.1

Exposure Time (ms)

0.439

Scan Rows

Scan Columns

FOV Height

2,032

FOV Width

2,428

Stain Type

Stitched Image

Image Name

C02533C1_SC_20240131_165052_3.0.3

Stitching

Template Source Row No.

The row number of the template FOV used for predicting the entire template

Template Source Column No.

The column number of the template FOV used for predicting the entire template

Global Height

Height of the stitched image

Global Width

Width of the stitched image

Template Source Row No.

Template Source Column No.

Global Height

22,151

Global Width

24,288

Image QC version

QC Pass

Whether the image(s) passed image QC quality check

Track Line Score

Reference score for track line detection

Clarity Score

Reference score for image clarity

Good FOV Count

Number of FOVs that have at least one track dot detected

Total FOV Count

Total number of FOVs

Stitching Score

Reference score for stitching

Tissue Segmentation Score

Reference score for tissue segmentation

Registration Score

Reference score for auto-aligning image with gene expression matrix

Image QC Version

3.0.3

QC Pass

Pass

Track Line Score

Clarity Score

-999

Good FOV Count

Total FOV Count

132

Stitching Score

Tissue Segmentation Score

100

Registration Score

Registration

ScaleX

The lateral scaling between image and template

ScaleY

The longitudinal scaling between image and template

Rotation

The rotation angle of the image relative to the template

Flip

Whether the image is flipped horizontally

Image X Offset

Offset between image and matrix in x direction

Image Y Offset

Offset between image and matrix in y direction

Counter Clockwise Rotation

Counter clockwise rotation angle

Manual ScaleX

The lateral scaling based on image center (manual-registration)

Manual ScaleY

The longitudinal scaling based on image center (manual-registration)

Manual Rotation

The rotation angle based on image center (manual-registration)

Matrix X Start

Gene expression matrix offset in x direction by DNB numbers

Matrix Y Start

Gene expression matrix offset in y direction by DNB numbers

Matrix Height

Gene expression matrix height

Matrix Width

Gene expression matrix width

ScaleX

0.996

ScaleY

0.997

Rotation

-0.385

Flip

True

Image X Offset

-316.49

Image Y Offset

4,108

Manual ScaleX

Manual ScaleY

Manual Rotation

Counter Clockwise Rotation

Matrix X Offset

0.00

Matrix Y Offset

0.00

Matrix Height

26,460

Matrix Width

26,460

Horizontal Image Stitching Deviation Heatmap

Image stitching deviation in the horizontal axis, a lighter color indicates better stitching (the heatmap and frame of axes have been adjusted with the registration parameters)

Vertical Image Stitching Deviation Heatmap

Image stitching deviation in the vertical axis, a lighter color indicates better stitching (the heatmap and frame of axes have been adjusted with the registration parameters)